Transfer and transformation characteristics of Zn and Cd in soil-rotation plant (Brassica napus L and Oryza sativa L) system and its influencing factors

Rice–rape rotation is a widely practiced cropping system in China. However, changes in soil properties and management could change the bioavailability of Cd, In order to explore the occurrence state, transportation and transformation characteristics of heavy metals Cd and Zn in rice-rape rotation system in Guizhou karst area with high background value of Cd. In the karst rice–rape rotation area, the physical and chemical properties of soil, chemical specifications and activities of Cd and Zn at different soil depths and during various crop growth stages, and the bioaccumulation of Cd and Zn in different tissues of rice and rape were studied by field experiment and laboratory analysis. The bioaccumulation of Cd and Zn and the effects of physical and chemical soil properties on the activities and bioavailabilities of Cd and Zn during rice–rape rotation were explored. The findings revealed that soil particle size, composition, pH, redox potential, soil organic matter, and Cd and Zn contents varied dramatically, especially in deep soils. The physical and chemical properties of the deep and surface soils were significantly related to the bioaccumulation of Cd and Zn. Cd and Zn are activated during crop rotation. Cd was easier to be enriched in rice, while Zn was easier to be enriched in rape. The correlation between Cd and Zn contents in Brassica campestris L and their enrichment abilities were not significant, but that in Oryza sativa L were significant. During rice-rape rotation, the chemical speciations and activities of Cd and Zn changed with the changes of soil properties and waterlogging environment. This study had important basic guiding significance for the evaluation, prevention and control of heavy metal pollution, and improving soil quality in different rotation systems in karst areas, and was conducive to promoting the safe production of rape and rice.

Sample collection and preparation. Samples of the surface soil (0-20 cm depth) and deep soil (20-40 cm depth) were collected before sowing and during different growth stages of the two crops (B. napus L: seedling stage, bolting stage, blooming period, and harvest period; O. sativa L: seedling stage, tillering stage, grain-filling period, and harvest period). Each sampling unit had five sampling points, and each sampling point was in a circular area with a radius of about 50-60 cm. The sampling points avoided rice field ridges, roads, etc. The sampling method for deep soil during flooding was as follows: a 20-40 cm soil column was collected using a stainless steel drill, and the outer layer of the soil that had touched the metal drill material was removed. Simultaneously, plant samples were collected in each soil sampling unit according to the soil sampling position, with the plant sampling points corresponding to the individual soil sampling points. Each sampling point was fixed, and continuous sampling was carried out in different growth stages, thus avoiding the error caused by the change of sampling points in the whole rotation process.
After the plant samples were brought to the laboratory, the tissues (roots, stems, leaves, flowers, etc.) were treated separately. The plant samples were washed with deionized water, dried at 75 °C ± 2 °C and ground to 0.25 mm. After the soil samples were air-dried in a cool and ventilated place, grinded and sifted to 2 mm, and then a part of the sample was taken by quartering method and continued grinding until the sample was sieved to 0.25 mm for analysis of physicochemical properties. www.nature.com/scientificreports/ Index analysis. Soil particle size analyses were performed using the hydrometer method 36 , and the Kaczynski system was used to classify the soil particles as follows 37 : fine clay (< 0.001 mm), coarse clay (0.001-0.005 mm), fine silt (0.005-0.01 mm), coarse silt (0.01-0.05 mm), fine sand (0.05-0.25 mm), and coarse sand (0.25-1 mm). Soil pH (soil-distilled water ratio of 1:2.5 w/v) and Eh (direct determination with platinum electrode) were measured using a mV/pH combined meter (Rex Electric Chemical PHS-3E, China) 36 . The SOM was digested by K 2 Cr 2 O 7 -H 2 SO 4 and examined using FeSO 4 titration 36 . For total metal analysis, the soil and plant samples were digested, respectively, using HNO 3 + HF (10:5 v/v) and HNO 3 + H 2 O 2 (10:5 v/v) and deionized water was maintained at a constant volume of 50 mL 36 . The Cd level was determined using a graphite furnace atomic absorption spectrometer (Agilent 240 Z, Agilent, Santa Clara, CA, USA), and Zn was determined using an inductively coupled plasma emission spectrometer (Leeman Prodigy XP, USA). Chemical speciation of Cd and Zn was determined using Tessier`s five-stage sequential extraction procedure 22 , and the supernatant extracted using the four-step method was tested using the same total metal analysis method. The content of RES was calculated using the subtraction method: where C total metal represents the total metal content, and C i represents the contents of chemical speciation fractions (EXC, CAR, IMO, and OM, respectively).
Accuracy was assessed using the sample spike and recovery method, and the average recoveries of Cd and Zn were 91.2% and 102.6%, respectively. The ratio of the sum of the five chemical speciation fraction concentrations of heavy metals in the soil samples to their total concentrations ranged from 78.63 to 125.84%.
The activities of heavy metals in the soil can be described using their movement factor (MF) 38 : where, C EXC and C CAR represent the concentrations of EXC and CAR, respectively, and ∑C five speciation represents the sum of the five chemical speciation fraction (EXC, CAR, IMO, OM, and RES) concentrations of the metal. The activation degrees of soil heavy metals were expressed using the activation coefficient (AD): where, MF soil-rice and MF soil-rape represent the average movement parameters of heavy metals in the soil throughout the rape and rice planting seasons, respectively. AD > 1 indicates that soil heavy metals are activated, whereas AD < 1 indicates that soil heavy metals are passivated. The bioconcentration factor (BCF) is the ratio of heavy metal content in plants to that in soil 7 . BCF represents the degree of migration of heavy metal elements in soil-plant systems and the heavy metal enrichment capacity of plants 1 . BCF is expressed using the following formula: where, BCF and T-BCF represent the bioenrichment capacities of different plant tissues and whole plants toward soil heavy metals, respectively. The C i , C plant , and C soil represent the contents of heavy metals in different aboveground tissue parts, whole plants of B. napus L and O. sativa L, and soil, respectively.
The biotranslocation factor (BTF) is an evaluation of the ability of plant tissues to transport heavy metals 39 : www.nature.com/scientificreports/ where, C root , C stem , C rapeseed , C rape pod , C rice husk , and C rice represent the heavy metal contents of the roots and stems of B. napus L or O. sativa L, rapeseed, rape pod, rice husk, and rice, respectively; C other tissues represents the heavy metal contents of the leaves, followers, rape pod, and rapeseed of B. napus L or the leaves, rice in the husk, rice husk, and rice of O. sativa L; and C aboveground parts represents the aboveground part of B. napus L or O. sativa L.
Statistical analysis. All data have been expressed as means and standard errors of three replicates. The data were subjected to analysis of variance using the SPSS, ver.21.0 statistical software package (SPSS, Chicago, IL, USA), and differences between the mean values were compared using the least significant difference post-hoc test. P values of < 0.05 were considered statistically significant. Correlation coefficients between the variables were tested using Pearson's correlation. Principal component analysis (PCA) and stepwise regression equations were used to evaluate the influencing factors of soil heavy metal activity and plant bioaccumulation. Graphs were constructed using OriginPro 9.0 (Origin Lab, Northampton, MA, USA).
Ethics statement. The collection of plant materials in this study complied with relevant institutional, national, and international guidelines and legislation.

Results
Soil properties. Soil particle size composition. As shown in Fig. 2 Overall, compared with surface soil, deep soil was more viscous, and the percentages of clay particles in the soil during the rice planting season were higher than those during the rape planting season. These differences were more obvious in deep soil (Fig. 2). In addition, the variation coefficients (cv) of the percentages of fine sand and fine clay were 31.41% and 18.88%, respectively, in surface soil (Table 1). However, in deep soil, fine sand (cv = 68.90%) and fine clay (cv = 30.01%) showed strong  www.nature.com/scientificreports/ variability, followed by coarse sand (cv = 24.39%) and fine silt (cv = 23.26%) ( Table 1). These results showed that after soil flooding, clay particles, fine silt particles, and other materials move vertically along the soil pores under the action of water gravity, causing the clay particles of paddy soil to move down, and the core soil layer is relatively viscous.
Soil pH. The soil in the study area was neutral to alkaline (Fig. 3a), which is typical for calcareous soil developed from carbonate rock. Throughout the rape planting season, the pH value of the surface soil ( Fig. 3a) did not change significantly and varied within a very narrow range (7.80-7.92). On the contrary, the pH value of deep soil changed significantly (6.9-8.07) and showed a downward trend that began during the pre-sowing period and extended into the blooming period. The pH rose again during the harvest period (Fig. 3a). Throughout the rice planting season, the trends in pH change were the same in surface and deep soil; in deep soil, it was higher (7.89-8.11) than that in surface soil (7.69-7.89) (Fig. 3a). In general, the change of soil pH in deep soil was greater than that in surface soil throughout the rape and rice planting season (Fig. 3a).
Soil Eh. The Eh of the soil was significantly affected by water level, soil depth, and the interactions of these factors (Fig. 3b). Eh values were positive throughout the rape planting season, and the Eh of surface soil (10.20-179.30 mV) and deep soil (29.2-193.3 mV) rose in a fluctuating pattern (Fig. 3b). While the Eh of surface soil and deep soil decreased sharply to negative values after rape harvest, the Eh of surface soil increased gradually during the rice seedling stage and did not differ much from that of deep soil at the tillering stage. Finally, both tended to be stable (Fig. 3b), and overall, an obvious variation was observed in the Eh of the soil during the flooding and drainage periods. Additionally, the Eh variations of surface soil were greater than those of deep soil (Fig. 3b).  www.nature.com/scientificreports/ SOM content. The SOM content was clearly affected by the alternation of drought and flood (Fig. 3c). The SOM of surface soil showed no significant change and was higher than that of deep soil, whereas the change in deep soil was obvious, especially during the rice planting season. The SOM content of deep soil before sowing rice decreased by 23.37 g/kg compared with that during the rape harvest period but increased slightly after the tillering stage (Fig. 3c).
Cd and Zn contents in the soil. As shown in Fig. 4a, Cd contents in both surface (0.48 mg/kg-1.81 mg/ kg) and deep soil (0.53 mg/kg-1.93 mg/kg) first increased and then decreased throughout the rape and rice planting season (except during rape pre-sowing). The content of Zn in surface soil (69.98 mg/kg-83.47 mg/kg) was higher than that in deep soil (68.37 mg/kg-80.82 mg/kg) and fluctuated greatly in surface soil during the rape planting season (Fig. 4b). The Zn/Cd ratio (Fig. 4c) showed little variation throughout the rape planting season until the rice tillering stage but increased significantly after that. There was no significant correlation between Cd and Zn in surface soil (Fig. 5a), but there was a significant negative correlation (r = − 0.569) in deep soil (Fig. 5b), indicating that Cd and Zn had obvious interaction inhibition (Fig. 5b).
Chemical speciation of Cd and Zn in the soil. The proportions of Cd chemical speciation fractions generally followed the sequence RES-Cd > IMO-Cd > CAR-Cd > OM-Cd > EXC-Cd (Fig. 6a) and Zn followed the sequence RES-Zn > IMO-Zn > OM-Zn > CAR-Zn > EXC-Zn (Fig. 6b). The changes in chemical speciation fractions of Cd and Zn were obvious during the rape-rice rotation. The five chemical speciation fractions of Cd in the surface soil fluctuated greatly, but the fluctuations of Zn were smaller. The fluctuations of Cd in deep soil were also smaller than those in surface soil, but those of Zn were greater. In surface soil, the percentages of EXC-Cd, CAR-Cd, OM-Cd, and IMO-Cd during the rice planting season were higher than those during the rape planting season (increasing by an average of 32.49%, 54.79%, 40.06%, and 37.7%, respectively), whereas the percentages of RES-Cd were the opposite (decreased by 13.88%), and among  (Fig. 7). The results also showed that the chemical speciation of Cd in the surface soil changed from RES-Cd to CAR-Cd, IMO-Cd, OM-Cd, and EXC-Cd. However, compared with surface soil, the percentages of the five chemical speciation fractions of Cd in deep soil varied to a lesser extent (Fig. 6a). Moreover, it is noteworthy that the percentages of RES-Cd and RES-Zn decreased significantly from the grain-filling period, whereas those of the other four fractions increased significantly (Fig. 6a,b). Similar to Cd, the proportions of EXC-Zn, CAR-Zn, IMO-Zn, and OM-Zn in surface soil increased during the rice planting season compared with those during the rape planting season (increasing by an average of 74.30%, 125.41%, 37.18%, and 4.51%, respectively) ( Fig. 6b), whereas the proportion of RES-Zn decreased by 9.00% (Fig. 6b). Likewise, the proportions of EXC-Zn and CRA-Zn in deep soil increased by 69.51% and 5.38%, respectively, whereas the changes in IMO-Zn and OM-Zn were opposite to those in surface soil (decreasing by 21.11% and 29.50%, respectively) ( Fig. 6b). There were significant correlations among the chemical speciation fractions of Zn (Fig. 7), among which EXC-Zn, CAR-Zn, and IMO-Zn in the surface soil showed significant positive correlations and the three fractions (EXC-Zn, CAR-Zn, and IMO-Zn) showed significant negative correlations with OM-Zn and RES-Zn. Additionally, there was a positive correlation between OM-Zn and RES-Zn (r = 0.527) (Fig. 7). On the contrary, in deep soil, EXC-Zn and RES-Zn were negatively correlated with the other three fractions, and there were significant positive correlations between EXC-Zn and RES-Zn (r = 0.576) and among CAR-Zn, IMO-Zn, and OM-Zn (r = 0.670, 0.698, and 0.934, respectively) ( Fig. 7).

Activation of the heavy metals Cd and Zn in the soil.
Although the content of Zn in the soil was significantly higher than that of Cd ( Fig. 4a,b) throughout the rape and rice planting season, the activity changes of Cd and Zn in surface soil and deep soil were not consistent. Specifically, the activities of Cd and Zn in surface soil increased significantly during the flooding process after the rape harvest and then remained stable during the rice planting season, which increased to 36.06-74.40% (Cd, Fig. 8a) and 87.75-172.79% (Zn, Fig. 8b). On the contrary, the activities in deep soil fluctuated slightly before the grain-filling period and then greatly increased (increased by 65.28% (Cd) and 104.90% (Zn)) ( Fig. 8a,b). In the dry-wet alternating process from rape planting season to rice planting season, the activation coefficients (AD) of the heavy metals Cd and Zn were both > 1 and were activated to varying degrees. The activation effect of Zn was slightly higher than that of Cd (Fig. 8d), but the activity of Cd in the soil was significantly higher than that of Zn ( Fig. 8a,b). This result further indicated that the activities of Cd and Zn were jointly expressed by their total contents and proportion of available chemical speciation fractions. Additionally, coexisting ions influenced each other's reactivity. Significant positive correla- Correlation analysis between the physical and chemical properties of the soil and the chemical speciation of heavy metals. As shown in Table 2, throughout the rape-rice planting season, coarse sand www.nature.com/scientificreports/ was significantly negatively correlated with the total content of Cd in both surface (r = − 0.547) and deep soils (r = − 0.700) and Eh exerted positive effects on the total contents of Cd and Zn, which were significant in surface soil. In deep soil, Cd content was significantly positively correlated with fine sand (r = 0.424) and Zn content was significantly positively correlated with SOM (r = 0.401). In surface soil, there was no obvious correlation between physical and chemical properties or with EXC-Cd and OM-Cd (Table 2). Eh (r = − 0.443) and fine sand (r = − 0.409) content had significant negative effects on CAR-Cd but had significant positive effects on RES-Cd. Coarse sand had significant positive effects on CAR-Cd (r = 0.480) and IMO-Cd (r = 0.391) but had a significant negative correlation with RES-Cd (r = − 0.826). pH had positive effects on RES-Cd (r = 0.587), OM-Zn (r = 0.408), and RES-Zn (r = 0.559) but was negatively correlated with CAR-Zn (r = − 0.382). EXC-Zn, CAR-Zn, and IMO-Zn were negatively correlated with fine sand and coarse silt but were significantly positively correlated with coarse sand (Table 2). In contrast, OM-Zn and RES-Zn were significantly positively correlated with fine sand and negatively correlated with coarse sand (Table 2).
In deep soil, except for the total content of Zn and EXC-Zn, pH was negatively correlated with the total content of Cd (r = − 0.335) and other chemical speciation fractions of Cd, CAR-Zn, IMO-Zn, OM-Zn, and RES-Zn (Table 2). Eh levels were negatively correlated with OM-Cd (r = − 0.154) and EXC-Zn (r = − 0.650) but were positively correlated with other chemical speciation fractions of Cd and Zn. SOM was significantly negatively correlated with EXC-Zn (r = − 0.640) but positively correlated with the chemical speciation fractions of Cd and Zn, all of which were significant, except for OM-Cd (Table 2). Besides the significant positive correlation with EXC-Zn (r = 0.604), fine clay was negatively correlated with Cd, Zn, and their chemical speciation fractions. Fine silt had a significant positive correlation with EXC-Zn (r = 0.494) and significant negative correlations with EXC-Cd, IMO-Cd, IMO-Zn, OM-Zn, and RES-Zn. Coarse silt had significant positive correlations with EXC-Cd, RES-Cd, IMO-Zn, and RES-Zn. Fine sand was positively correlated with RES-Cd, IMO-Zn, OM-Zn, and RES-Zn but negatively correlated with EXC-Zn. Coarse sand was negatively correlated with RES-Cd and positively correlated with OM-Cd and CAR-Zn.
Soil is a complex system, and its chemical properties frequently interact with each other to jointly affect the chemical speciation fractions of heavy metals in it. The activities of heavy metals are directly reflected by their total content and chemical speciation fractions.
Step-up linear regression analyses indicated that CAR was the primary factor affecting the activity of Cd and Zn, followed by EXC, both of which had positive effects on the activity of Cd and Zn (MF-Cd and MF-Zn) ( Table 3). On the contrary, Eh was the primary soil chemical property limiting the activities of heavy metals both in surface and deep soil (Table 3). Additionally, MF-Cd and MF-Zn were limited by their residual fractions in the surface soil. While MF-Cd was limited by fine silt, fine sand had a positive effect on it (Table 3). Meanwhile, pH and fine silt in deep soil had positive effects on MF-Cd and MF-Zn (Table 3), SOM had a positive effect on MF-Cd, and fine sand had a positive effect on MF-Zn (Table 3). However, MF-Cd was limited by IMO-Cd, and MF-Zn was limited by RES-Zn (Table 3).   10a) were significantly higher than those in other parts of the plant (P < 0.05), and Cd was easily transferred from stems to leaves (BTF = 1.99-2.11) (Table 4). Therefore, leaves were the primary enrichment organs for Cd. During the harvest period, Cd contents were higher in rape pods (0.42 mg/kg) and stems (0.39 mg/kg) and BCF of Cd (26.8% and 25.0%, respectively) was also significantly higher than that in roots (17.5%) and rapeseed (8.6%) (P < 0.05) (Fig. 10a). The content of Cd in roots (0.27-0.64 mg/kg) and stems (0.35-0.51 mg/kg) increased first and then decreased during different rape planting periods. After the bolting stage, Cd transport from stems to roots gradually increased (Table 4), which resulted in higher Cd content in stems (0.51 mg/kg, 0.39 mg/kg) than in roots (0.45 mg/kg, 0.27 mg/kg) during the blooming and harvest periods (Fig. 9a). Compared with other parts, the contents of Cd in flowers (0.12 mg/kg) and rapeseed (0.13 mg/kg) were the lowest (Fig. 9a) and the BTF from stems to flowers (BTF = 0.25) was the lowest (Table 4); hence, flowers were not the primary enrichment organ for Cd. Although the transport capacity of Cd from stems to rapeseed (BTF = 1.07) was higher than to rape pod (BTF = 0.34), the content of Cd in rapeseed (0.13 mg/kg) was still significantly lower than that in rape pod (0.42 mg/kg) ( Table 4). This result might be due to the short rapeseed forming period and the difficulty in transferring Cd from rape pod to rapeseed (BTF = 0.32).

Bioaccumulation of Cd and Zn in
In contrast, the contents of Zn in flowers (49.31 mg/kg) and rapeseed (47.50 mg/kg) were significantly higher than those in other plant parts (P < 0.05) (Fig. 9c), and the BCF of flowers (69.51%) and rapeseed (57.82%) were significantly higher than those in other parts (Fig. 10c). This finding indicated that flowers and rapeseed were the primary enrichment organs for Zn in B. napus L. In addition to leaf drop at harvest time, the contents of Zn in leaves (26.24-33.66 mg/kg) (Fig. 9c) were also high during other growth periods and were one of the primary organs for Zn enrichment, with the exception of flowers and rapeseed. During the vegetative growth period, Zn was primarily transferred from stems to leaves (BTF: 1.17-2.56) and then gradually to flowers (BTF: 4.01) and rape pod (BTF: 6.76) and rape seed (BTF:1.02) during the blooming period (Table 4). www.nature.com/scientificreports/ The variation trends in T-BCF of Cd (77.98-125.94%) and Zn (84.38-154.79%) in the whole plant were consistent (Fig. 10a-c) and were the greatest during the blooming period and the smallest during the harvest period. The overall variation range of Zn was greater than that of Cd. The T-BTF of Zn from roots to the aboveground parts was lower than that of Cd during the vegetative growth period but was significantly higher than that of Cd during the reproductive growth period ( Table 4).

Bioaccumulation of Cd and Zn in
Oryza sativa L at different growth periods. The contents of Cd were the highest in the roots (0.8-1.5 mg/kg) during different growth periods (Fig. 9b). The content and BCF of Cd in the roots increased gradually with the growth of O. sativa L, and the BCFs were > 1 during the grain-filling period (249.9%) and harvest period (315.8%) (Figs. 9b, 10b). Moreover, the BTFs of Cd from roots to stems were only 0.06-0.07 (Table 4), thereby resulting in most of the Cd being absorbed by and then fixed in the roots, which became the primary organ for Cd enrichment. At different growth periods, both the contents (0.19-0.21 mg/ kg) and BCFs (10.25%-39.39%) of Cd in leaves were higher than those in stems (Cd: 0.06-0.09 mg/kg; BCF: 3.32%-19.58%) (Figs. 9b and 10b) and the BTFs (2.01-3.17) from stems to leaves were all > 1 (Table 4). Hence, Cd was primarily enriched in leaves after upward transport from stems. The BCF of grain increased slightly from the grain-filling period to the harvest period, and the BTFs of Cd from stems to grain (1.59-1.83) were > 1 (Table 4). Therefore, the content of Cd in grain during the harvest period was higher than that during the grain- Table 2. Pearson's correlation coefficients of soil physical and chemical properties with the contents and five chemical speciation fractions of Cd and Zn. "*" indicate significant at P < 0.05.

Soil depth
Physical and chemical indexes of soil Heavy metal content and chemical speciation states Cd   www.nature.com/scientificreports/ filling period. Additionally, the BTF of stem to rice husk (0.83) was lower than that to rice (1.00) ( Table 4), and Cd from rice husk was continuously transferred to rice (1.21) (Table 4), which resulted in higher BCF of rice than that of rice husk, leaves, and stems (Fig. 10b). Similarly, during different growth periods of O. sativa L, Zn contents in roots were 21.59 mg/kg-32.79 mg/kg (Fig. 9d) and BCFs were 28.45%-43.59% (Fig. 10d). Except for the harvest period, the contents of Zn and BCF in roots were higher than those in other plant parts (Figs. 9d and 10d), and the BTFs (0.21-0.57) of Zn from roots to stems were low (Table 4), resulting in most of the absorbed Zn being trapped in the roots (Fig. 9d). The BTFs (0.99-2.60) of Zn from stems to leaves were greater (Table 4), and the BCFs of Zn in leaves (16.82-22.63%) were higher than those in stems (6.55-20.31%) (Fig. 9d). Therefore, leaves were also an important part for Zn enrichment. The contents and BCF of Zn in stems decreased first and then increased, and the lowest and highest levels were observed during the grain tillering period and seedling stage, respectively. The BCF of grain (rice in the husk) increased significantly from the grain-filling period (26.86%) to the harvest period (34.55%) (Fig. 10d), and the BTFs were 2.11-2.25. This led to the contents of Zn in grain (26.21 mg/kg) being the highest during the harvest period, and the Zn content in rice (16.56 mg/kg) was higher than that in husk (9.66 mg/kg).

EXC-Cd CAR-Cd IMO-Cd OM-Cd RES-Cd Zn EXC-Zn CAR-Zn IMO-Zn OM-Zn RES-Zn
The bioconcentration of Cd and Zn in O. sativa L was seen primarily in the roots and leaves. Additionally, the T-BCF of Cd (57.68-410.50%) in the whole plant was much higher than that of Zn (84.11-103.05%) (Fig. 10b,d). However, the BTF changes for Cd and Zn in all organs were the same. The T-BTFs of Cd (0.24-0.41) in aboveground parts of O. sativa were significantly lower than those of Zn (0.77-3.59) (Table 4). Furthermore, the BTFs of Cd from stems to leaves (2.01-3.17) were greater than those of Zn (0.99-2.60), but the BTFs of Cd from roots to stems (0.06-0.07) were lower than those for Zn (0.21-0.57), and the ability of rice to transport Zn (1.34-1.71) was also higher than that of Cd (1.00-1.20). These results indicated that although the O. sativa L roots could absorb Cd more easily, the transport capacity of Zn from the roots to the aboveground parts of the plant was stronger.
Influencing factors of Cd and Zn bioaccumulation during rape and rice rotation. The direct factors affecting the bioaccumulation of heavy metals in plants were their activities in soil and the abilities of plants  www.nature.com/scientificreports/ to accumulate them. The physical and chemical properties of soil indirectly affected the uptake of heavy metals by plants as a result of affecting the activity of heavy metals in soil. PCA analysis showed that there were significant differences in physical and chemical soil properties and heavy metal migration and transformation in different plants (O. sativa L and B. napus L) and plant growth periods during crop rotation (Fig. 11). During the entire rotation process, Eh, contents and activities of Cd and Zn, and the ratio of Zn to Cd content in soil had significant effects on the total Cd bioconcentration factor (Cd-BCF) and total Cd contents in plants (Plant-Cd). Among them, the contents of Cd, Eh, and coarse sand in soils of different depths had negative correlations with Cd-BCF and Plant-Cd (Fig. 11). The content and activity of Zn, SOM, fine sand, and coarse silt in deep soil (MF-Zn-II, SOM-II, fine sand-II, and coarse silt-II) had positive effects on the total bioconcentration factor of Zn (Zn-BCF) and the total Zn contents of plants (Plant-Zn). However, clay and pH in the deep soil (Clay-II, pH-II) and clay and MF-Zn in the surface soil had significant negative correlations with Zn-BCF and Plant-Zn (Fig. 11). Additionally, the bioconcentrations and contents of Cd and Zn in plants were positively correlated (Fig. 11). However, during the entire rape-rice rotation process, owing to differences in plant species (O. sativa L and B. napus L) and flooding conditions (alternating drought and flood), the influencing factors of heavy metal bioaccumulation of the two crops were different. Therefore, further stepwise linear regression simulation analysis was conducted on the influencing factors of O. sativa L, and B. napus L bioaccumulation (Table 5). These results indicated that deep soil had more significant effects on Cd bioaccumulation, whereas the effects of surface soil on Zn bioaccumulation was more significant during rape growth.
The SOM-II and ratio of Zn to Cd content in deep soil (T-Zn/Cd-II) had significant positive effects on Plant-Cd and Cd-BCF of B. napus L, whereas the ratio of Zn to Cd content in surface soil (T-Zn/Cd) had a significant negative effect (Table 5). Additionally, the ratio of Zn to Cd activity in deep soil (A-Zn/Cd-II) had a significant negative correlation with Cd-BCF of B. napus L ( Table 5) www.nature.com/scientificreports/ that Soil-Cd-II promoted Zn-BCF of B. napus L, whereas MF-Zn was significantly negatively correlated with Zn-BCF (Table 5). Moreover, MF-Cd had no significant effect on the Cd bioaccumulation of B. napus L, but the Zn/Cd ratio in soil had a significant effect (Table 5). During the process of O. sativa L growth, the influencing factors of Plant-Cd and Cd-BCF were somewhat different. The Plant-Cd was not only positively correlated with Cd-BCF, Soil-Cd-II, and Soil-Zn-II but also negatively correlated with MF-Zn and fine sand-II. While the Cd-BCF of O. sativa L was significantly negatively correlated with Soil-Cd, it was significantly positively correlated with MF-Zn-II and T-Zn/Cd (Table 5).
Zn-BCF of O. sativa L, Soil-Zn-II, pH-II, and SOM-II had significant positive effects on Plant-Zn of O. sativa L ( Table 5). The Zn-BCF of O. sativa L was significantly positively correlated with SOM-II and fine sand-II but negatively correlated with Soil-Zn and Soil-Cd-II (Table 5).
The Zn/Cd ratio in soil was found to have significant effects on Cd-BCF of B. napus L and O. sativa L. Furthermore, the Cd content in soil was also significantly correlated with Zn-BCF of B. napus L and O. sativa L, but the mechanism was affected by soil depth (Table 5).

Discussion
Bioaccumulation capacity of Cd and Zn in the soil by B. napus L and O. sativa L was different. The absorption and transport pathways of heavy metals in plants include active absorption and passive absorption by roots, transfer from stems to other parts of the plant. Heavy metals in stem, leaves and inflorescence are reactivated and transported into grain through phloem during flowering to seed maturity stage 40 . Additionally, plants transport Cd directly into seeds through xylem 41,42 . Therefore, the enrichment capacity of heavy metals was seen to increase during the grain development period. In this study, it was also confirmed that Cd and Zn absorbed by the roots of B. napus L and O. sativa L before the reproductive stage were mostly transported to the leaves and then transferred to the seeds during the reproductive stage. However, the migration and accumulation of elements varies in different plants. In rapeseed, the transport capacity of Cd was seen to be higher than that of Zn during the vegetative growth stage but lower than that during the reproductive stage (Table 4). Flowers and grains were the main enrichment organs for Zn, whereas their Cd content was the lowest (Fig. 9). Moreover, Zn was more easily translocated to rice grains than Cd (Table 4) probably because Zn is an important component of many enzymes and can promote the synthesis of carbohydrates and proteins. Therefore, the translocation of Zn to flowers and grains during the reproductive period is beneficial and increases the protein content of grains 43,44 . Furthermore, Zn enrichment may inhibit the transfer of Cd into seeds 29,45,46 .
This study found that although Cd was more easily absorbed by O. sativa L roots, Zn was more easily transported upward (Table 4). Cd and Zn enrichment capacities of B. napus L and O. sativa L were different. The enrichment ability of Cd in O. sativa L was higher than that in B. napus L but was the opposite for Zn. Conversely, the enrichment capacity of Zn in B. napus L was higher than that of Cd but was the opposite in O. sativa L (Fig. 9). It has been confirmed that heavy metal accumulation capacity varies widely among different varieties and species and that it is affected by the levels of heavy metals in the soil 32 . This study also found that the BCF of Cd and Zn in B. napus L was not the key factor affecting the contents of Cd and Zn in plants. The physical and chemical soil properties and the Zn/Cd ratio were the primary factors affecting the content in plants (Table 5). This result contrasts with the contents of Cd and Zn in O. sativa L, which were closely related to their BCFs (Table 5), and it is consistent with the findings of previous studies 39,47 . In addition to different metal properties, the affinity of different plants and the influence of physical and chemical soil properties on Cd and Zn were seen to be related. Some studies have found that O. sativa L is a unique crop with strong Cd accumulation capacity and significant Zn exclusion 48,49 . Interestingly, the rhizosphere exudates of B. napus L activate soil available cadmium to varying degrees, which have an impact on the cadmium content of rice grains in subsequent plantings 50 . In contrast, a reducing environment with low REDOX potential was formed due to the flooding environment in the paddy field, and hydrogen sulfide produced by the anaerobic decomposition of organic matter forms cadmium sulfide precipitates with Cd in the soil, thus reducing its availability 26 .

Soil-Zn/Cd ratio was closely related to the bioenrichment of Cd and Zn in rape and rice during crop rotation.
In soil systems, the interactions between heavy metals primarily occur at the substrate, absorption, and target levels, and there is site competition for heavy metals at all three levels 51 . Cd stress leads to severe Cd exposure and Zn deficiency in rice grains 44 , and high Cd/Zn ratios in rice could exacerbate the potential biotoxicity of Cd 23 . However, several studies have shown that the Cd/Zn ratio of crops inhibits Cd absorption to a certain extent 31,32,35 . The possible interaction between Cd and Zn in soil adsorption and plant absorption 32,42,44,45,52 is also one of the reasons for the difference in Cd and Zn enrichment between B. napus L and O. sativa. In this study, the bioenrichment of Cd in rape was closely related to the Zn/Cd ratio in soil at different depths. The Zn/Cd ratio in topsoil inhibited the absorption of Cd in rape, but promoted the enrichment of Cd in rice (Table 5). This may be related to the significant increase of Zn/Cd in soil during rice planting period. However, the correlation between Zn/Cd ratio in soil and Zn bioenrichment in rapeseed and rice was low. When Cd toxicity predominates, the presence of Zn antagonizes cadmium and reduces the toxicity of Cd to organisms. On the contrary, when Zn toxicity predominates, the presence of Cd has a synergistic effect with zinc and increases the toxicity of Zn to organisms 35,53 . Effects of soil physicochemical properties on the bioavailability of Cd and Zn during rice-rape rotation. Soil Eh. The change in Eh reflected the redox state of soil. Soil Eh decreased gradually from rape planting season to rice planting season and was in a reducing state. This observation was consistent with the results of previous studies where Eh was noted to decrease during the flooding process and increase during the drying process 54,55 . The Eh of surface soil should be higher owing to better soil aeration. However, good aeration www.nature.com/scientificreports/ is also more conducive to the growth of aerobic microorganisms, which consume oxygen during the growth process and in turn reduce the soil Eh 36 . This may be the reason for the Eh of surface soil being slightly lower than that of deeper soil during the rape planting period. In this study, Eh did not directly affect the bioavailability of Cd and Zn (Table 4) but did so indirectly by affecting the activities of these heavy metals in the soil. The degree of toxicity of the chemical forms of heavy metals is EXC > CAR > IMO > OM > RES 23 , and this study found that there was a significant positive correlation between soil Eh and the RES of heavy metals (P < 0.01) ( Table 2). In surface soil, the contents of CAR-Cd, CAR-Zn, EXC-Zn, and IMO-Zn increased significantly with the decrease in Eh, whereas the contents of OM-Zn decreased significantly. In deep soil, EXC-Zn and OM-Cd increased with the decrease in Eh (Fig. 6, Table 2). Therefore, Eh reduction was found to be one of the causes for heavy metal activation (Fig. 8a,b,d). Chuan et al. 56 also observed that when the Eh of soil solution decreased, the concentration of exchangeable Cd increased. Therefore, Eh determines the solubility change of heavy metals in the process of rice field-upland field rotation 25,57 .
In soil, redox substances, such as iron and manganese oxides and sulfides, indirectly affect the solubility and morphology of heavy metals in the soil [28][29][30] . When Eh decreases (such as in flooded environments), reductive substances increase in soil, a large number of iron and manganese oxides are reduced and dissolved, and previously stable adsorbed metal ions are released into soil solution 27 . Moreover, reduced Fe 2+ and Mn 2+ may compete with heavy metal ions and lead to their release 27 , thus enhancing the migration of heavy metal ions. Similar results were observed in this study where the percentages of residual fractions of Cd and Zn decreased with the decrease in Eh (Fig. 6) ( Table 2) and the activities increased ( Fig. 8a-b). However, the correlations between Eh and the IMO of metals in different soil depths were different. This observation may be related to the different redox environments due to the degree of change resulting from surface soil flooding and drying more than did deeper soil and the difference in pH values and SOM (Fig. 3). It is also possible that the adsorption capacity of different iron oxides to heavy metals is different (the adsorption capacities were in the following order: amorphous iron oxides > maghemites > lepidocrocite > goethite) 58,59 . With the decrease in Eh + pH, iron oxides change from amorphous forms with strong adsorption ability to microcrystalline forms with weak adsorption ability 60 . Therefore, Eh can change the speciation of iron and manganese oxides, and thus, affect the environmental behavior of heavy metals.
Soil pH. The effect of Eh on the activity of heavy metals is primarily through the influence of soil pH or through the interaction with pH change 61 . Soil pH changes in response to the change in Eh, and different H + activities directly affect the chemical species and the migration and transformation processes of heavy metals 62 . In this study, the carbonate binding fractions of Cd and Zn were the main factors that enhanced their activity, whereas the residual fractions were the main forms that limited their activity (Table 3). pH had negative correlations with Cd and Zn in the bonded fraction of carbonate and the oxidized fraction of iron-manganese and significant positive correlations with Cd and Zn in the residue fraction (Table 2). These correlations were closely related to the fact that the soil in the test area was neutral to alkaline (pH = 6.9-8.11) (Fig. 3a), which is a typical calcareous soil associated with carbonate rock. In general, most heavy metals in calcareous soil with pH > 7 are converted into carbonate fractions, and the increase in pH is conducive to the formation of carbonate 63 . However, some studies have found that Cd was likely to be hydrolyzed into Cd(OH) 2 or Cd(OH) + at pH 6.0-8.0 64 . In waterlogged environment, CdHCO 3 and Cd(OH) 2 CO 3 can be formed by hydrolysis of metal elements in the carbonate binding state, resulting in alkaline water and increased pH. In addition, the change of Eh is the main factor affecting the activity of heavy metals during crop rotation, and pH value is affected by Eh. It was found that soil pH tended to be neutral after flooding. Wang et al. 65 showed that when pH was 6.0-8.5, it continued to decrease as Eh decreased because of the decomposition of organic matter into a variety of small organic acids and carbon dioxide. In this study, there was a slight change in pH under flooding condition. It may be that a large amount of carbonate hydrolysis in the soil where carbonate rocks developed led to the increase of pH, which played a buffering role in pH change under flooding condition 66 . Therefore, in this study, owing to the complexity of soil composition, there is not a single positive/negative correlation between Eh and pH 67 , and under the comprehensive influence of complex changes in soil chemical environment, pH changes are negatively correlated with Cd and Zn in the bonded fraction of carbonate. However, the specific mechanism of the effect of pH value on the change of carbonate in the dry-wet alternating environment needs further investigation.
In addition, Cd(OH) + and Zn(OH) + formed by the hydrolysis of Cd and Zn in the bonded fraction of carbonate have strong affinity and can be adsorbed on the surface and lattice of soil minerals via electrostatic adsorption, ion exchange, and hydrogen bonding 68 . In alkaline environments, iron and manganese oxides are mostly negatively charged, and the adsorption of heavy metals increases rapidly with the increase in pH 69 . Therefore, heavy metals primarily exist in the form of compounds in alkaline soil, which may be the reason for the low activity of heavy metals in soil observed in this study (MF-Cd: 6.51-12.31%; MF-Zn: 0.48-1.90%).
It is noteworthy that in this study, pH had no significant effect on Cd and Zn activities in surface soil but had a positive and significant effect in deep soil (Table 3). This observation may be because the pH of deep soil was more sensitive to the influence of heavy metal speciation than that of surface soil, and as can be inferred from Fig. 3a, the pH increased with soil depth. This was consistent with the distribution rule of pH values in different soil depths 70,71 .
Soil organic matters. In addition to pH, Eh may also affect the composition of organic matter 72 . SOM possesses complex organic functional groups that can adsorb, complexate, or chelate metal ions 73 . Moreover, SOM has strong reducibility, which can reduce highly-charged heavy metals and alter their toxicity 74,75 . In this study, the variation range of organic matter in the surface soil was low, whereas the variation degree in deep soil was more obvious, especially after rapeseed harvest, which was significantly reduced during the flooding process (Fig. 3c). www.nature.com/scientificreports/ When flooded in deep soil in an anaerobic environment for a long time, macromolecular organic matter easily acts as an electron acceptor through microbial reduction into dissolved organic matter (DOM) 76 . Meanwhile, metals bound to organic matter are released as the organic matter decomposes and then combine with DOM 77 , thereby resulting in increased metal concentrations in soil solutions in overlying water and increased availability 78,79 . This is one of the main reasons for the observed increase in soil metal (Cd and Zn) activities during rice planting season and also for the bioaccumulation of metals in B. napus L and O. sativa L being significantly affected by SOM (Table 5). Zhao et al. also showed that organic matter increases the bioavailability of Cd in rice 80 . The SOM increase in deep soil during the harvest stage may be due to the reduced nutrient requirements of plants and the increased amount of organic matter in deep soil due to senescent roots. Accordingly, soil DOM content also increases and competes with heavy metals for adsorption sites on soil surfaces, thus reducing the adsorption capacity of soil for heavy metals and increasing the exchangeable metals ions. Consequently, the activities of Cd and Zn in deep soil increased to varying degrees (Fig. 8a). Additionally, drainage during the later stages of O. sativa L growth altered the redox environment of the underlying soil, and the mineralization and decomposition of organic matter also released metal ions.
Soil particle sizes. Soil texture has a major impact on soil adsorption and affects the distribution and bioavailability of heavy metals 81 . In general, smaller soil particles have better adsorption properties and, therefore, higher heavy metal concentrations 82,83 . This study reached a similar conclusion that heavy metals were primarily concentrated in particles sized 0.05-0.25 mm. During the rice planting period, the proportional increase in small soil particles (Fig. 2) and the percentage of clay (< 0.01 mm) in the soil were negatively correlated with the bioavailability of heavy metals (Fig. 11). This finding indicates that water caused certain mechanical dispersion forces that affected the soil particles, reduced the soil particle size, increased the adsorption of heavy metals, and reduced the availability of Cd and Zn. These effects were far lower than the effective enhancement of heavy metals by redox environment changes caused by water logging. Hence, the activity of heavy metals continued to increase after waterlogging (Fig. 8). Soil particle size generally does not directly affect the chemical species of heavy metals but affects the concentration of heavy metals in the soil solution through their absorption-desorption by soil particle size components. Thus, the combination, complexation, or chelation of heavy metals with organic substances, clay minerals, and other organic and inorganic colloids is affected 82,83 . Consequently, the distribution proportions of heavy metals in soil grains affect their toxicity 84 .
In conclusion, the frequent interaction of soil phases makes the activity of each relative heavy metal extremely complicated, and the difference of heavy metal enrichment of rotating plants leads to the difficulty of controlling heavy metals under rotation mode. Eh is an important variable affecting the migration of heavy metals in soil of rotation system, while Fe, Mn, and sulfide in soil components are essential media, and soil microorganisms are also crucial to the chemical form of heavy metals. Therefore, the above factors should be considered in the study of heavy metal release in soil with Eh fluctuations, so as to better understand and predict the dynamic change mechanism of heavy metals under the influence of complex alternating dry and wet effects. In addition, the dynamic changes of heavy metals in the rotation system under spatial heterogeneity and time scale, as well as the quantitative relationship between soil environment and heavy metal activity in the process of rapeseed-rice rotation will be further studied.

Conclusion
This study found that the physical and chemical properties of soil and heavy metal migration and transformation processes were significantly different during the growth periods of different plants (B. napus L and O. sativa L). Soil-Cd content varied greatly at different depths, and Zn content in surface soil fluctuated greatly compared with that in deep soil. Due to the change of soil physical and chemical properties, the chemical species and activities of Cd and Zn changed significantly and were activated obviously during the rice growth season, in which Zn chemical species and activity were more easily affected. In the rotation process, the enrichment ability of Cd in O. sativa L was stronger, while that of Zn in B. napus L was stronger, and the transport characteristics of Cd and Zn in different organs of the two crops were also different. Cd and Zn contents in the two crops were mainly affected by the physical and chemical properties of the soil and Soil-Zn/Cd ratio, whereas that of O. sativa L was also closely related to the enrichment ability. Zn and Cd exhibited certain interactions in soil-plant systems, especially in deep soil. Eh was the main factor affecting the chemical properties and activities of soil heavy metals, but it was not a direct factor affecting the bioenrichment of O. sativa L and B. napus L. However, the changes in soil SOM, pH, and grain size affected the bioenrichment ability of B. napus L. In conclusion, alternating dry and wet cropping changed the activities of Cd and Zn, soil properties, and heavy metal activity, which differed during different growing stages of crops. The absorption capacity of different crops toward different heavy metals also varied. Therefore, corresponding mitigation actions should be taken according to the specific soil environment and plant characteristics involved in the treatment process.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding authors on a reasonable request. www.nature.com/scientificreports/